The present invention relates to a method of measuring a spectral response of a biological sample. In particular, the invention relates to a method of measuring the spectral response by irradiating the sample with broadband probe light and sensing spectral changes of the probe light, which result from an interaction of the probe light with the sample, like a method of measuring absorption and/or reflection of the probe light at the sample. Furthermore, the invention relates to a spectroscopic measuring apparatus for measuring a spectral response of a biological sample, in particular including a broadband light source for irradiating the sample with probe light and a detector device for spectrally resolved detecting changes of the probe light resulting from an interaction of the probe light with the sample. Applications of the invention are available in spectroscopy of samples, in particular for analysing a composition and/or condition of a sample. Biological samples, which can be analysed comprise e.g. samples from a human or animal organism or samples taken from a natural environment.
For illustrating background art relating to techniques for analysing substance samples, in particular biological samples for diagnostic purposes, reference is made to the following prior art documents:    [1] B. de Lacy Costello et al., “A review of the volatiles from the healthy human body”, J. Breath Res. 8, 014001 (2014);    [2] T. H. Risby et al., “Current status of clinical breath analysis”, Appl. Phys. B 85, 421-426 (2006);    [3] W. Cao et al., “Breath analysis: Potential for clinical diagnosis and exposure assessment”, Clinical Chemistry 52, 800-811 (2006);    [4] US 2012/0266653 A1;    [5] U.S. Pat. No. 7,101,340 B1;    [6] WO 2011/117572 A1;    [7] U.S. Pat. No. 5,222,495;    [8] U.S. Pat. No. 8,022,366 B2;    [9] U.S. Pat. No. 6,236,047 B1;    [10] U.S. Pat. No. 7,403,805 B2;    [11] U.S. Pat. Non. 7,203,345 B2;    [12] EP 0,680,273 B1;    [13] P. Dumas et al., “Adding synchrotron radiation to infrared microspectroscopy: what's new in biomedical applications?” TRENDS in Biotechnology 25, 40 (2006);    [14] I. Znakovskaya et al., “Dual frequency comb spectroscopy with a single laser”, Opt. Lett. 39, 5471 (2014);    [15] A. Sponring et al., “Release of volatile organic compounds from the lung cancer cell line NCI-H2087 In Vitro”, Anticancer Research 29, 419 (2009);    [16] M. Diem et al., “Molecular pathology via IR and Raman spectral imaging”, J. Biophoton. 6, 855 (2013);    [17] W. Parz et al., “Time-domain spectroscopy of mid-infrared quantum cascade lasers”, Semicond. Sci. Technol. 26 (2011) 014020;    [18] WO 2007/121598 A1;    [19] US 2013/0221222 A1;    [20] B. Bernhardt et al., “Mid-infrared dual-comb spectroscopy with 2.4 μm Cr2+:ZnSe femtosecond lasers”, Appl. Phys. B (2010) 3; and    [21] Sh. Liu et al., “Mid-infrared time-domain spectroscopy system with carrier-envelope phase stabilization”, Appl. Phys. Lett. 103, 181111 (2013).
In medicine there is an urgent need for a minimally invasive, rapid, reliable, and cost-effective diagnosis of diseases at early stages (screening) and for monitoring their response to therapy. It is generally known that the analysis of biological samples, including body fluids and gases emitted from the body is well suited for this purpose because they contain a multitude of compounds characteristic of the health status of a person. About 1760 different such components are known, specifically 874 in exhaled breath, 504 in skin emanations, 279 in urine headspace, 130 in blood, 381 in feces and 353 in saliva [1]. Importantly, some compounds exist only in the liquid phase some in both gas and liquid phases. In particular, breath aerosol is potentially rich of heavy compounds.
Any change in the structure of molecular constituents of a human cell invariably causes a change in the mid-infrared (MIR) absorption spectrum of the cell itself or of its metabolic emanations. As a consequence, small modifications in the spectrum offer a means of early detection and diagnosis of many diseases. The statistically-proven spectral traces of a disease will provide reliable “fingerprint” information for its early diagnosis.
In classical diagnostics, the compounds of biologicals samples are detected by chemical analysis or by gas chromatography combined with mass spectrometry [2, 3]. However, these methods i) do not allow for fast analysis, ii) can modify or even destroy some compounds and iii) are blind for conformational changes in the structure of DNA, which may, without any change in mass, initiate severe diseases.
Furthermore, a number of spectroscopic methods have been suggested for the examination of body fluids and gases [4-12, 16]. In [4] gas analysis alone is proposed whereas actually all phases (gas, liquid, solid, aerosol) can contribute to diagnostic knowledge. In [5] spectral analysis of breath by cw lasers is suggested and thus the number of available spectral data points and their informative value is very limited. In [6] a narrow range of wavelengths is used and only three gases are detected, restricting the range of diagnostics to diabetes.
Further conventional approaches dealing with the spectral analysis of body liquids, such as blood or saliva, are described in [7] and [8]. Patent [7] proposes non-invasive blood analysis by comparing the absorption of two closely spaced wavelengths in blood. In [8] a compact MIR spectrometer is proposed for measuring blood sugar (glucose) and other blood and body fluid analytes. It consists of a modulated thermal emitter and a low-resolution spectrometer containing quarter wave plates acting as interference filters.
Diffusively reflected radiation of bands in the range of 1100 to 5000 nm is used in [9] to determine concentrations of blood analytes by chemometric techniques. Another technique uses a contact device placed on the eye to investigate spectral changes in the conjunctiva and the tear film [10]. Thermal radiation from the eye itself or external radiation supplied by a fiber are employed for this purpose. Spectroscopy is used in [11] to identify individuals by analysing the reflection of near-infrared radiation from human tissue. In [12] a catheter containing a fiberoptic bundle is inserted into gastro-intestinal compartments for the detection of fluorescence and absorption of light by their contents.
As a general disadvantage, none of the conventional methods is capable of providing the full information on the health status of a person that would in principle be available. The conventional techniques are specialized for using a single phase for diagnosis only. Furthermore, they employ only a narrow spectral range within the full MIR-bandwidth, and they are not sensitive enough to detect subtle changes in the spectrum indicative of a disease. In other words, the known approaches offer access only to a small fraction of the full Molecular fingerprint and even that with a sensitivity and signal-to-noise ratio that is insufficient for reliable identification and diagnosis of diseases.
Recently the use of synchrotron radiation has been explored for spectroscopic imaging of cells with various kinds of disorders [13]. This radiation is broadband and about two orders of magnitude more intense than that of a thermal source. However, the application of synchrotrons for routine diagnostics and for screening a large number of patients does not appear practical.
The above limitations do not occur with analysing biological samples for diagnostic purposes only. Other spectroscopic investigations, e. g. of environmental samples or laser media, have similar disadvantages, in particular in terms of sensitivity, selectivity and limited use of available information.
As an example, [17] discloses a spectroscopic investigation of a quantum-cascade-laser (QCL). For measuring gain and absorption, the QCL is irradiated with 10 fs laser pulses having a wavelength in the MIR range, and the spectroscopic response of the QCL is investigated using a time domain spectroscopy setup with electro-optic detection. The application of the conventional method is restricted to the investigation of strong absorbing QCL materials. Due to the use of a Ti-sapphire laser, the laser pulses have a low intensity, so that measurements of weak absorptions are excluded. Furthermore, the laser pulses have a narrowband characteristic, resulting in limitations for investigating other materials with spectral features in a broad wavelength range.
Another application of a Ti-sapphire laser for creating THz radiation in a range of 1.3 to 4.8 THz, corresponding to a wavelength in a range of 62 μm to 230 μm, by optical rectification in an organic material is disclosed in [18]. According to [19], a narrow frequency range around 6 μm is investigated using a Ti-sapphire laser. Due to the narrow wavelength ranges, the low radiation intensity and a limited stability of the laser setup, these conventional technique are not suitable for an efficient spectral broadband characterization of materials.
Dual-comb spectroscopy for investigating a gas sample is described in [20]. This method is restricted to an FTIR measurement in a narrow wavelength range between 2.3 μm and 2.6 μm, reaching only a low sensitivity in a ppm-range. Again, this technique is not suitable for investigating materials with spectral features in a broad wavelength range.
MIR-radiation in a range from 8 μm to 12 μm can be created on the basis of Er:fibre laser emissions at three wavelengths of 1050 nm, 1350 nm and 1550 nm as disclosed in [21]. This technique requires a complex loop control, resulting in restricted applicability of the MIR-radiation.
A first objective of the invention is to provide an improved method of measuring a spectral response of a sample, which is capable of avoiding limitations or disadvantages of conventional techniques. In particular, it is the first objective of the invention to provide the measuring method with an increased sensitivity, improved signal-to-noise-ratio (SNR), enhanced selectivity and/or improved capability of covering an extended spectral range, e. g. in the mid-infrared spectral range (MIR). A second objective of the invention is to provide an improved spectroscopic measuring apparatus, which is adapted for measuring a spectral response of a sample to a probe light irradiation, wherein the spectroscopic measuring apparatus is capable of avoiding limitations and disadvantages of conventional techniques. In particular, the spectroscopic measuring apparatus is to be capable of providing improvements in terms of sensitivity, SNR, selectivity and/or broadband coverage.
These objectives are correspondingly solved by a spectral response measuring method and a spectroscopic measuring apparatus of the invention.